Enzyme-antibody conjugates are used in a number of immuno-assay techniques based on ligand-antiligand technology. The more common techniques are enzyme linked immunosorbent assays (ELISA), enzyme linked oligonucleotide immunosorbent assays (ELOSA) and immunoblotting. The antibodies most frequently used in the techniques are of the IgG and IgM type.
Customarily, the preparation of useful conjugates for immuno-assays involves the reaction of antibody with an excess of enzyme in order to achieve the most efficient and complete conjugation of antibody to enzyme. As a result of using excess enzyme, the conjugate reaction mixture will necessarily contain free, unconjugated enzyme. The free enzyme serves no beneficial purpose in immuno-assays and, in fact, causes nonspecific staining, called background staining.
Historically, methods which have been used to eliminate the presence of free enzyme after conjugate formation have not been totally satisfactory. Methods involving the use of extreme conditions, such as pH, can destroy or severely damage the enzyme part of the conjugate. More gentle procedures such as gel filtration are not generally applicable when large aggregates of enzyme are created, such as when glutaraldehyde is used to couple enzyme to antibody. When the enzyme of choice is alkaline phosphatase, a large and popular enzyme, purification by gel filtration presents particularly special problems.